Transient Assay for Promoter Activity of Metallothionein-Like Gene from Oil Palm in Driving Tissue-Specific Expression of Polyhydroxybutyrate and Reporter Genes
Omidvar, Vahid (2006) Transient Assay for Promoter Activity of Metallothionein-Like Gene from Oil Palm in Driving Tissue-Specific Expression of Polyhydroxybutyrate and Reporter Genes. Masters thesis, Universiti Putra Malaysia.
Polyhydroxybutyrate is the most important type of polyhydroxyalkanoates (PHA), which is naturally synthesized by several microorganisms. Production of PHB in plants is based on the consumption of acetyl-CoA as an initial substrate, therefore oil palm being an oil crop serve as potential target due to its high flux of acetyl-CoA during the oil synthesis stage. In oil palm, mesocarp is the specialized tissue for oil synthesis. Targeting the expression of PHB genes in mesocarp tissue requires the mesocarp-specific regulation of these genes by a mesocarp-specific promoter, since accumulation of PHB in other tissues may have deleterious effects on the plant. Pervious efforts have resulted in isolation of promoter sequence of the oil palm mesocarp-specific metallothionein-like gene (designated MSP1) and preparation of the gene construct carrying PHB genes (pMS29 vector) to drive the specific expression of PHB genes (phbA, phbB, and phbC) in mesocarp tissue. Analysis of the specificity and strength of MSP1 and evaluating the expression pattern of the PHB genes in oil palm mesocarp tissue in a transient expression system will provide insight information prior to the stable transformation and supports time-saving strategy and more targeted and precise use of the technique. In this study, expression of PHB genes under the regulation of MSP1 was evaluated in a biolistic-based transient assay in transiently transformed oil palm mesocarp and leaf (control) tissue slices. Transcriptional and translational analysis of PHB genes was carried out by Reverse-transcriptase PCR (RT-PCR) and western blot techniques. RT-PCR analysis revealed that the engineering of PHB biosynthetic pathway genes under the regulation of MSP1 in transformed mesocarp tissue has resulted in successful transcription of phbA and phbB genes; however the phbC gene failed to produce any transcription product. In addition, RT-PCR analysis in non-transformed (non-bombarded) mesocarp tissues showed that there is no endogenous expression of PHB genes, indicating that the expression of PHB genes in transformed mesocarp tissues is the result of the PHB pathway engineering. Furthermore, no expression level of PHB genes was detected in leaf tissue slices after bombardment. Western blot analysis using polyclonal antibodies specific for phbB and phbC genes in transformed mesocarp tissues confirmed the successful translation of phbB mRNA transcript into protein product, however clearly revealed the failure in translation of phbC gene. Western blot analysis of phbB and phbC gene products in transformed leaf tissues revealed that regulation by MSP1 did not result in translation of these genes. Further analysis in non-transformed tissues showed that phbB and phbC gene products do not exist in mesocarp and leaf tissues prior to the transformation. As a conclusion, although engineering of PHB biosynthetic pathway genes in mesocarp tissue did not result in an entirely functional pathway, it resulted in transcription of phbA and phbB genes and successful translation of phbB gene. This demonstrated that bacterial genes irrespective of their source and functions can be transcribed and translated in plant tissues under the regulation of plant tissue-specific promoters. The tissue-specificity and strength of MSP1 was evaluated via transient reporter assays of GUS and GFP reporter genes in different oil palm tissues, including mesocarp, leaf, and root. The constitutive CaMV35S promoter was used as a reference in all analysis. Histochemical GUS assay showed the expression of GUS driven by MSP1 in transformed mesocarp tissue slices, while no expression of GUS gene was detected in transformed leaf and root tissue slices. Using the CaMV35S promoter, GUS expression was observed strongly in all transformed mesocarp, leaf, and root tissue slices. Quantitative analysis of GFP driven by MSP1 in transformed tissues revealed that GFP was expressed dominantly in mesocarp tissue slices (2.7 times more than it was expressed in leaf, and 86 times more than root), however there was some expression level of GFP directed by MSP1 in transformed leaf and root tissues. Comparative analysis of GFP expression, driven by the CaMV35S and MSP1 promoters showed that the CaMV35S promoter has the stronger activity with the average of 1.4 times more than the activity of MSP1 in transformed mesocarp tissue. This result indicated that although MSP1 is a strong promoter and has a great tendency to up-regulate the gene expression in mesocarp tissue, but the promoter does not behave in a hundred percent tissue-specific manner.
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